Modular automated chromatography system

ABSTRACT

Valves, pumps, detectors, sample loops, fraction collectors and the like are individually incorporated into modules that are mountable at individual mounting sites on a base unit which also supports one or more chromatography columns. Each module includes fluid connections to other modules and a microcontroller joining the module to a computed and monitor through an electronic connector at each mounting site. The fluid connections between the modules and the column(s) are removed from the electronic connections and accessible to the user. A software platform may recognize the modules and their locations, coordinate fluid connections between the modules, and provide a variety of control, monitoring, data generating and data processing functions to generate chromatographic data. The software platform may also provide graphical tools for designing chromatographic methods from a library of phases.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.15/071,138, filed Mar. 15, 2016 and titled “Modular AutomatedChromatography System”, which is a continuation of U.S. patentapplication Ser. No. 13/591,461, filed Aug. 22, 2012 and titled “ModularAutomated Chromatography System”, which claims the benefit of U.S.Provisional Patent Application No. 61/526,959, filed Aug. 24, 2011 andtitled “Modular Automated Chromatography System”, the contents of whichare hereby incorporated by reference herein by reference in theirentirety.

BACKGROUND OF THE INVENTION Field of the Invention

This invention resides in the field of automated chromatography systems.

Description of the Prior Art

The extraction and purification of proteins, peptides, nucleic acids,monoclonal antibodies, and other species of interest from biologicalsamples, as well as from reaction mixtures and fluid media in general,are critical procedures for research laboratories, diagnosticlaboratories, drug manufacturing operations, and any number of settingsand locations in the biotechnology industry. These procedures havereached such a high level of sophistication that virtually anybiological species can be obtained in a highly purified form. Eachspecies has its own special needs however in terms of such factors assample preparation, the type and composition of separation media, andthe conditions under which separation is performed. To meet all of itspurification needs, therefore, the typical laboratory must accommodate avariety of components, protocols, and materials. Flexibility andadaptability are needed but are often obtained at the expense ofconsistency and accuracy, and the ability to accommodate a variety ofprotocols and to do so with the requisite accuracy often entails highcosts in equipment, labor, and training.

SUMMARY OF THE INVENTION

The present invention addresses this need in a system that is easy andinexpensive to operate and that is highly adaptable to the separationand purification of different species and the protocols needed for eachspecies. The system is easily and quickly assembled, reconfigured,substituted, and expanded, and, when the system is in use, its operationis readily monitored, controlled, and maintained. Even with complex flowschemes, the fluidics lines of the system are accessible to the user andmanaged in a neat and logical manner, while the electrical connectionsare confined to locations apart from those of the fluidics lines and arehidden from view. Most, if not all, of the electrical connections can beachieved through sockets, although certain embodiments of the inventioninclude cables for some or all of the electrical connections.

According to one aspect, a system for joining a plurality of fluidmanipulation components into a flow scheme for directing fluids to andfrom a chromatographic separation device and for operating said joinedfluid manipulation components according to a selected protocol comprisesa plurality of modules, each module comprising (a) one of said fluidmanipulation components, and (b) a microcontroller that transmitsoperational signals to said fluid manipulation component in response tocommands received from outside said module. The system further comprisesa mounting frame comprising a plurality of bays, each of the pluralityof bays constructed to receive a single module. At least some of themodules are constructed to receive single modules interchangeably withother said modules. Each of the plurality of bays comprises a signalconnector that couples to and communicates with the microcontroller of amodule mounted in said bay. The system further comprises a softwareplatform in communication with the microcontroller of each module, saidsoftware being programmed to (1) receive from each of the plurality ofmodules information identifying a type of fluid manipulation componentin said module, (2) recognize from the received information the types ofthe fluid manipulation components in the plurality of modules, (3)automatically map the plurality of fluid manipulation components to theflow scheme based on the received data, (4) provide to a user of thesystem visual indications where fluid connections to the plurality ofmodules are needed in order to implement said flow scheme using theplurality of modules, and (5) cause said fluid manipulation components,once joined, to direct fluids to and from said chromatographicseparation device in accordance with said protocol.

According to another aspect, a computer system for configuring andcontrolling a chromatography system comprises a graphical display, aninput device, a communication interface, a processor, and software. Whenexecuted, the software causes the computer system to receive aspecification of a chromatography flow scheme having a number of fluidconnections, receive via the communication interface data from a numberof components of the chromatography system, the data identifying andindicating a type of each of the components, map the identified fluidmanipulation components to the chromatography flow scheme; and display arepresentation of the flow scheme on the graphical display. Each of thecomponents is mounted in a respective mounting site of a mounting frame,each mounting site comprising a signal connector that couples to thecomponent and through which the commands are communicated from thecomputer system to the component.

Systems in accordance with the present invention can thus accommodate awide range of separation media and fluid transfer components. Thefeatures and embodiments cited above, together with other features andembodiments of the invention, are described in detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is perspective view of a modular chromatography system inaccordance with the present invention with components separated for easeof viewing.

FIG. 1B is a view of one module of the system of FIG. 1A, shown inperspective from the rear.

FIG. 2 is a front view of a base unit in accordance with the inventionas an alternative to the base unit of the system of FIG. 1A.

FIG. 3 is a diagram of a system in accordance with the present inventionwith multiple workstations.

FIG. 4 is an outline for a computer program for use in the invention.

FIG. 5 is a screen shot, or graphical display, of one flow scheme thatcan be displayed on a monitor in accordance with the invention.

FIG. 6 is a screen shot, or graphical display, of a second flow schemethat can be displayed on a monitor in accordance with the invention.

DETAILED DESCRIPTION OF THE INVENTION AND SELECTED EMBODIMENTS

Modules. The term “module” is used herein to denote one or morefunctional devices affixed to a plate, an enclosure, or a supportingstructure in general that is of standardized shape and size, thestandardization enabling the module to be received, inserted, orotherwise affixed to any of a plurality of sites on the mounting frame.The standardized construction of the modules also enables each moduleupon mounting to the frame to engage the central user interface andsoftware through couplings that join the microcontrollers on the modulesto the signal connectors in the frame. As noted above, each modulecontains at least one fluid manipulation component and at least onemicrocontroller, plus any other devices necessary for the operation ofthe fluid manipulation device, and mounting features that mate withcomplementary mounting features at sites on the mounting frame. In avalve module, for example, the fluid manipulation device will be avalve, and the module can also contain a motor or actuating device asneeded to rotate or actuate the valve. Actuating or operationalsupporting devices for any other type of fluid manipulation componentwill be readily apparent to those skilled in chromatographic and fluidflow equipment. The mounting structures on the modules can be tabs,pegs, hooks, latches, flanges, or anything for which a complementarystructure can be built into the frame such as a slot, a shelf, a groove,a contoured recess or opening, for a secure mounting.

Fluid manipulation components. The term “fluid manipulation component”is used herein to denote components through which fluid passes orcomponents that direct the flow of fluid in a particular direction.Examples of functions that a fluid manipulation component might performare: driving the flow of fluids, combining or dividing fluids, detectingfluids, analyzing fluids, and measuring fluids. Examples of fluidmanipulation components are: valves, pumps, mixers, sample loops,detectors, and fraction collectors. The valves can be simple shut-offvalves or multi-port valves including rotary valves; the pumps can berotary pumps, peristaltic pumps, syringe-type pumps, gradient pumps, orother laboratory-scale pumps; the detectors can be single-wavelength UVdetectors, UV-Vis detectors, conductivity detectors, pH detectors,refractive index detectors, fluorescence detectors, pressure detectors,temperature detectors, or air or gas sensors; and the mixers can includeproportioning mixers, either constant or variable.

Microcontrollers. The microcontrollers that are embedded in each modulecan be conventional integrated circuits, each containing a processorcore, a memory, and programmable input/output peripherals. Themicrocontroller encodes machine-readable data which can include theidentity of the component contained in the module, for example, as wellas operational characteristics and features of the component, andreal-time operational status of the component. The data can thus bereadable by the system to inform the software of the identity of thecomponent, i.e., whether the component is a valve, a detector, or any ofthe other types of components listed above, whether the component iscurrently operating or on standby, the flow rate through the componentif operating, temperature, pressure, position the case of a valve, andthe like. The microcontroller can also transmit operational signals tothe component in response to commands received from a computer that iseither incorporated into or external to the mounting frame. In certainembodiments, the microcontroller also detects malfunctions of the fluidmanipulation component, such as those indicated by a rising pressure, arising temperature, or a failure to respond to a command. Themicrocontroller in these embodiments will then emit an alarm signalalerting the user or the system software of the malfunction. An alarmindicator can be included on an exposed surface of the module, and thealarm can be a visually observable change in the indicator, such as alight turning on. Microcontrollers in certain embodiments of theinvention can contain or be coupled to radio-frequency identification(“RFID”) tags that can be updated by the user or by the module itself tocontain information regarding current status of the fluid manipulationcomponent or accumulated data pertaining to its use.

Component status indicators. An optional feature for inclusion in amodule is a display that indicates the operational status of thecomponent contained in the module on a real time basis. The statusinformation, which will be readable by the user and specific to thecomponent, can indicate for example the position of a valve, the flowrate through a valve or a pump, the direction of a pump, the action of adetector in detecting species in solution or a change in condition, thepressure in a pump, and any such indicator from which the user canvisually detect whether the component is operating normally or isexperiencing malfunction. The status indicator can be any device thattransmits a visual or otherwise detectable signal, that can be mountedto the exterior surface of the module, and that can display the statusinformation in a user-readable form, such as a numeric or graphical formor simply by an indicator light. A liquid crystal display (LCD) is oneexample of such an indicator; other examples will be readily apparent tothose of skill in the art.

Mounting sites and mounting frame; modular and interchangeable characterof modules. The mounting sites are thus complementary to the modulesboth in their mounting features and in the locations of the signalconnectors in the mounting sites relative to the microcontrollers on themodules, such that any module can be mounted to any of differentmounting sites. Examples of mounting sites are slots, cavities,indentations, brackets, bays, or any such structure that will support amodule in a secure and fixed position to minimize or avoid thepossibility of disengagement through jostling of, or inadvertent impactsto, the module or the mounting frame. A simple example is a mountingsite designed for a module to be slid into position at the site. Themounting frame is generally a chassis or structure with mounting membersat designated sites on the structure that serve as mounting sites, andcan thus be a support rack with cavities, bays, or open spaces ingeneral for insertion of the modules. Most conveniently, the mountingsites can be spatially arranged in a regular geometric pattern, such asrows, columns, or a two-dimensional grid of rows and columns. Certainmodules can be designed to occupy two or more adjacent mounting sites,and thus for example the mounting frame can include bays with widths orheights that are multiples of those of other bays, such as toaccommodate double-width and triple-width, or double-height andtriple-height, modules in addition to modules that occupy singlemounting sites. Such doubling or tripling can be achieved for example byconstructing the mounting sites as bays that are separated by verticalor horizontal barriers that are removable to combine adjacent bays. Instill further embodiments, the mounting frame can be constructed toallow expansion of its module capacity by the addition of further rowsor columns of mounting sites that can be supplied as accessories to themounting frame.

In certain embodiments of the invention in which the mounting sites arearranged in two or more rows, the rows are fixed relative to each other,with all mounting sites accessible from one side of the mounting frame.The components of the various modules in these embodiments will besubstantially coplanar, i.e., in substantially a common vertical planeat the front of the mounting frame. In many cases, this will improve theease of making fluidic connections between the components mounted to theframe through their modules. In other embodiments of the invention wherethe mounting sites are arranged in two or more rows, one or more of theupper rows are secured to the underlying rows in a swiveling orrotatable manner, allowing the upper row(s) to be rotated relative tothe row(s) beneath, the axis of rotation being vertical axis and passingthrough the center of the frame. In certain flow schemes, this rotationcan improve access to or from certain fluid manipulation components.Fluid reservoirs that are placed to one side of the mounting frame, forexample, can be connected to valves, pumps, meters, and other componentson the frame with shorter lengths of tubing, and a column mounted to oneside of the frame can likewise benefit from shorter tubing connectionsto pumps, detectors, or the like mounted to one of the mounting sites.Shorter tubing lengths will minimize the mixing of species, fractions,and liquids in general between the mounted components, where such mixingmight lessen the resolution between bands of analytes, lower the slopesof pH or salt gradients, or lessen the sharpness of a stepwise change influid composition when such a change is part of a protocol. In somecases, the rotation capability may require longer tubing linesconnecting some of the components. Certain modules, for example, mayhave fluid manipulation components that protrude from the frame when themodules are mounted in one of the mounting bays and extend above, below,or to one side of the projected dimensions of a single bay, hinderingthe placement of modules with a similarly protruding components inneighboring bays. Rotation of one row of bays in these cases will allowthe modules to be mounted without interfering with each other. Onearrangement that will accommodate these and other needs is a mountingframe with four rows of mounting bays, the two uppermost rows beingrotatable relative to the two lower non-rotatable rows, andindependently of each other. For safety, the rotatable rows in certainembodiments can be automatically locked in position when electricalpower is supplied to the mounting frame and released for rotation onlywhen the power is off.

Column rack. The fluid manipulation component on one or more modules canbe a chromatographic column. Chromatographic columns can also (oralternatively) be positioned and/or supported separately from themounting frame with appropriate fluidic connections to the components inthe modules. A further alternative is to mount such columns to themounting frame at sites separate from the module mounting sites, sincethe columns will typically be able to function without beinginterchangeable with the fluid manipulation components in a flow scheme.The column mounting sites can be included in a column rack at locationson the mounting frame that are apart from the module mounting sites andthat do not interfere with fluidic or operator access to the modules andtheir components. Thus, with the module mounting sites on a front faceof the mounting frame and the electrical connections at the back face ofthe mounting frame, the column rack can be on one of the side faces andyet accessible to the modules on the front face for fluid tubingconnections to the modules. The rack can be designed to accommodateeither a single column or two or more such columns, and in the lattercase one or more of the rack positions can be left vacant occupied atthe option of the user, mounting only the number of columns that areneeded for a selected flow scheme. Information pertaining to the columnson the rack can be incorporated into the data used and displayed in thesystem by including identifier tags on the columns that are readable bythe system software, and in certain cases, that are capable of havinginformation added to them by the user through the system software.Examples of such tags are radio-frequency identification (“RFID”) tags;other examples will be readily apparent to those of skill in the art.The information contained in a single tag can include the type ofcolumn, such as for example affinity, ion-exchange, hydrophobicinteraction, gel filtration, or isoelectric focusing. The informationcan also include the column volume, the separation medium in the column,and the history of use of the column, such as the number of separationsperformed in the medium, the pressure the column experienced when lastused, the last date at which the column was cleaned in place and theconditions using in the cleaning, and the results of the last columnperformance test. To read and utilize these tags, the rack can containsensors appropriate to the particular tags, and the informationtransmitted by the sensors to the system software can include thelocation on the rack at which the column has been mounted in addition toall of the other information contained in the tag.

Software platform. Embedded in the mounting frame in certain embodimentsof this invention is a server containing a suite of firmware, i.e.,fixed programs or data structure to support the communication with andoperation of the microcontrollers, and one or more libraries ofoperating programs for individual fluid manipulation devices or forpre-established fluidics schemes, or both. The mounting frame in manyembodiments is also equipped with a touch screen monitor, preferably onemeasuring ten inches (25.4 cm) or greater. The touch screen monitor canserve as a GUI (graphical user interface) and can be equipped with USB(universal serial bus) ports for data import or export, ports for akeyboard and mouse, and jacks for LAN (local area network) or Ethernetcables. Alternatively, the mounting frame can include a mounting sitefor a tablet PC such as an Apple iPad or any such device that has aninstalled application that allows the device to function as the touchscreen. Alternatively to, or in conjunction with, the touch screenmonitor or tablet PC, a standard desktop personal computer with aconventional operating system such as Microsoft WINDOWS can be used foroperation and control. When both a touch screen monitor mounted directlyto the mounting frame and a desktop computer external to the mountingframe are used, the two can be connected through an LAN, an Ethernet, orany other standard communication protocol, either wired or wireless.

Certain embodiments of the invention also contain programmable softwareembedded in either the mounting frame or the desktop computer. Thissoftware can be programmed to interrogate each microcontroller in thevarious modules for the machine-readable data encoded in themicrocontroller, and to do so immediately upon the mounting of eachmodule to the mounting frame. The microcontroller can respond to theinterrogation by sending a signal back to the system software, and thesoftware can correlate the signal with an embedded library to determinethe type and/or identity of the component in the module. The responsefrom the microcontroller can also contain instructions for the commandsthat the module requires for operation. The software will then recordthe identity and location of the component and display this informationon the GUI in the form of icons with the position of each icon on theGUI representing the location of the corresponding component on themounting frame. One library for example can contain pre-designed flowschemes and component combinations from which the user can select aparticular flow scheme or portion of a flow scheme. A further librarycan contain additional icons for components to be added to a flow schemeat the choice of the user, in a “drag-and-drop” manner. If a fluidicsscheme has been selected from an embedded library in the monitor or thedesktop computer, or has been devised by the user, the GUI can show thetubing connections needed to form the desired fluidics scheme.

Software can also be included for guiding the user to properly installfluid lines, i.e., tubing connections, between the various modules, andoptionally to inform the user of any incomplete or missing connections.For proper functioning of the entire system, all of the proper fluidconnections must be made, a process known as “plumbing” the system. Whendone manually, the making of such connections is a time-consumingprocess and prone to error. In certain embodiments of this invention,the plumbing process is simplified and the risk of error is reduced byinclusion of a “click-and-plumb” function in the software. Such afunction can identify the proper connection sites for each fluid lineone at a time and display the connection sites in a manner that willallow the fluid connections to be made in sequence.

The array of connections representing the flow scheme for the entiresystem can be displayed as a diagram of the system, showing the route offluid flow between modules in a graphical format. A tubing connectionbetween two modules within the graphical representation can be “clicked”(i.e., by a mouse or otherwise) to cause the system to turn on indicatorlights on the modules themselves near the two locations between which atubing connection should be installed. Once the user installs the tubingconnection, another click by the user can turn off the indicator lightsfor that connection and turn on two different lights representing thenext connection. Any type of light indicator can be used; of these, LEDsare often the most convenient. Alternatives to lights being turned on oroff are lights changing color; indeed, any visual clues can be used, aswill be apparent to the skilled software engineer. This click-and-plumbprocess can continue until all of the tubing connections are made. Thesystem is then fully plumbed and ready for operation.

An alternative to using LEDs or other indicator lights on the modulesfor the click-and-plumb process is to use computer graphics that show adiagram or picture of the flow scheme as a display on a computer monitoror touch screen, the display containing virtual indicator lights thatcorrespond to each of the sites for the tubing connections. Theclick-and-plumb process is performed by the individual in an analogousmanner, except that the virtual indicator lights, rather than lights onthe modules themselves, will turn on to indicate the connections to bemade and then off to indicate that they have indeed been made. Certainembodiments will include both a display with virtual indicator lightsand actual indicator lights on the modules, offering the user a choicebetween the two. Tubing connections can also be shown by means otherthan, or in addition to, indicator lights. The system diagram may thusshow the connection points but not the tubing until each tubingconnection is made, in which case the appearance of a line connectingtwo points on the diagram is an indication that a successful and correctconnection has been made between the connections corresponding to thosepoints. Alternatives to the absence/appearance of the line representingthe tubing connection are a change in color, a change from highlightingto absence of highlighting (or vice versa), a change from normal lineintensity to a bold line (or vice versa), and a change from a blinkingline to a steady line (or vice versa). Other alternatives will bereadily apparent to those of skill in computer graphics.

The “click-and-plumb” function can be initiated by conventional softwaremeans, such as a selection on the system control screen or a menuselection. The active fluidics scheme described above can be replaced bya wizard interface and the selection of a “next” function, as theseterms are known in computer software. Other equivalent means will bereadily apparent to those of skill in the art. The “click-and-plumb”function can be operated through a touch screen affixed to the system orthrough a computer in proximity to the system, with a button or switchon the system to initiate and advance the function. The button or switchis particularly useful when the system is retained in a refrigeratedenvironment and the computer is placed outside the environment.

As an alternative to the “click” portion of the function, the system canbe programmed to automatically advance between plumbing locations atfixed time intervals. Sensors can also be installed on the system todetect tubing connections that have been made. Examples of such sensorsare optical sensors, electrical sensors, and pneumatic sensors. The“click-and-plumb” function can also be used for checking that all fluidconnections have been properly made. To do so, the system can identifythe correct connections sequentially to allow the user to check anddetermine whether a tubing line has in fact been installed to make eachconnection. As in the installation function, the display of individualconnections can be advanced manually or automatically. All of thesefunctions can be performed either with indicator lights on the modulesor with virtual indicator lights or tubing connection indicators on amonitor or touch screen display.

The overall software platform, whether embedded in the mounting frame orin a desktop computer or distributed between the mounting frame and adesktop computer, will also contain programs dedicated to operating thefluidics scheme hardware, including causing the fluid manipulationcomponents to direct fluids to and from the chromatographic separationcolumn by way of the selected flow scheme. This function will forexample include energizing or de-energizing pumps, opening, closing, orrotating valves, and similar operations to implement protocols forprocessing samples and operating the column. When the mounting framesoftware includes a GUI, the GUI can also include demonstrationprotocols that can be run both with and without attachment to theinstrument, i.e., with and without any components having been engaged,to show the user how the system operates and how it generates andanalyzes a chromatogram. In certain cases as well, the GUI will containhelp files to assist the user in the setup and calibration of theinstrument, the reading of component specifications, the design andprogramming of fluidics schemes and protocols, and troubleshooting.

As mentioned above, a single fluidics system consisting of modulesmounted to a mounting frame equipped with a touch screen monitor can betermed a “workstation,” and two or more such workstations can beindependently operated and controlled by a common desktop computerexternal to the mounting frame or base unit. Each workstation can bepowered independently, or a power distribution unit can be used withports for multiple workstations. In either case, workstations can beadded to or subtracted from the desktop computer without the need foradditional wiring.

Software suite in external computer. When a desktop or other externalcomputer is used, it can include a software suite that includes majorchromatography functions and that can be configured to operate at anyscale from an analytical or laboratory scale to a preparative scale,with additional functions appropriate to the scale of operation.Components of the software suite can include administration software,method editor or programming software, system control software, andevaluation software. Examples of functions that can be performed bythese software components are as follows.

Administration software. Administration software can include amulti-level access system for different levels of authority and control.As an illustration, the access system can have five levels includingadministrator access as Level 1, supervisor access as Level 2, scientistaccess as Level 3, operator access as Level 4, and restricted useraccess as Level 5. When multiple workstations are connected to a singleexternal computer, an administrator can use administrator access to nameand create users for each workstation, or supervisors who will in turnname and control users through the supervisor access. Through the useraccess, a user can then view all workstations on which that user hasbeen designated, and select a particular workstation on which a run isto be performed. Scientist and restricted user access can be used toestablish other levels of access and use. Data generated by aworkstation can be stored in a database management system which iseither on the same computer in which the software suite is loaded or ona remote or external computer owned by the user and accessed through SQL(structured query language) or other such authentication. Databases ineither the workstation or the external computer can contain data tablesthat can be extended, modified, and rapidly sorted through so that thedata can be rapidly retrieved, exported, and backed up as desired.

An optional feature of the administration software is an ability toestablish audit trails and electronic signatures for compliance with 21CFR Part 11 regulations as promulgated by the Food and DrugAdministration and published in the Federal Register of Mar. 20, 1997,Vol. 62, No. 54. Further optional features, of particular interest inindustrial environments subject to regulatory review, are the inclusionof verification and validation protocols in the form of IQ/OQ/PQ(installation qualification, operational qualifications, performancequalifications) procedures, and the ability to generate a final reportsummarizing and referencing all protocols, results, and conclusions.

Method editor software. With this software component, a user, whether anindividual or a group of individuals, can create a method either througha project folder or through a start menu. Methods can be created bysequencing groups of steps referred to as phases, using a pre-programmedmethod template. A method can also be created by modifying an existingmethod. Regardless of how a method is created, the software can includea prompt to the user offering the option to save the method prior tostarting a run. A user creating such a method will be able to define themethod settings, such as the parameters to be used for that method and amethod outline. These method parameters and results can then be saved ina database where they are linked to the method from which they aregenerated. Parameters of the method can include the following:

-   -   Chromatography mode, examples of which are anion exchange,        cation exchange, mixed-mode ion exchange, size exclusion,        hydrophobic interaction, affinity, and isoelectric focusing    -   Column parameters and requirements, examples of which are        physical dimensions, pressure limits, flow rates, connector        types, and part numbers    -   Operational limits, examples of which are maximum pressure and        minimum flow rate    -   Selected operational parameters, for example the means of        expressing elution flow volumes or rates, such as by number of        column volumes, linear velocity, volumetric velocity, or simply        time; buffer blending to achieve dilution, pH adjustment, and pH        or concentration gradients

The method outline will allow the user to create a sequence of phases,which are groups of logical steps that are common to chromatographyprocedures in general. Examples of such steps are equilibration, sampleapplication, column activation, column washing, elution, columnpreparation, system preparation, and column performance tests. As notedabove, these phases can be stored in a library, and the selection ofphases or of groups of phases from the library, and the assemblage ofselected phases or groups into a chromatographic protocol, can beperformed by the user through the GUI. Each phase will have its owndefault operational parameters, modifiable by the user. Parameters foran equilibration phase, for example, can include flow rate, buffer saltconcentration, buffer gradient, equilibration time, and buffer volume.Parameters for sample application can include manual application,application through a sample pump, application either through a sampleloop or directly onto the column, and application through anautosampler, and protocols for washing the sample loop and for fractioncollection, which can include parameters for fraction differentiation,for the means of fraction separation, for fraction destinations, and forfraction detectors. Parameters for column washing can include flow ratesand the selection, concentration, gradient, and amounts of wash bufferto be used. Parameters for elution can include choices between isocraticelution, linear gradients, stepwise gradients, and gradient curves.Parameters for column preparation can include the choice ofequilibration solution, the volume of solution to be used, the solutionflow rate, and the incubation time. Parameters for system preparationcan include selection of buffers, concentrations and quantities forpriming the system tubing and inlets with buffer solution and the choiceof inlets, outlets, column positions to be prepared, and incubationtime. Parameters for a column performance test can include a choice ofblank (non-absorbing) liquid or solution to pass through the column andthe condition of the column during the passage of the liquid orsolution, to determine such factors as height equivalent to atheoretical plate (HETP) and peak asymmetry factor (As). Further phasesfor possible inclusion can include column clean-in-place and systemclean-in-place functions, for removing residual or non-specificallybound species without removing the column from the column rack or fromthe tubing connections between it and the modules.

The method editor software can contain pre-programmed methods suppliedwith the instrument, which can serve as templates for modification bythe user. Examples are methods of system preparation, columnclean-in-place methods, system clean-in-place methods, columnperformance tests, column preparation, column desalting, and methodsspecifically designed for particular chromatographic techniques such asaffinity chromatography, anion exchange chromatography, cation exchangechromatography, chromatofocusing, gel filtration (size differentiation),hydrophobic interaction chromatography, and reverse-phasechromatography.

The method editor software can also include scouting functions, by whichthe system systematically varies one parameter of a phase and runs thevariations in sequence to determine the optimal value of that parameterfor a particular procedure. Systematic variations of this type areuseful in screening columns, identifying an optimal pH, identifying amaximum sample volume for a particular column, identifying an optimalflow rate for a particular column, and identifying an optimum gradientlength, slope, or stepwise configuration. Scouting parameters to beselected by the user can include column volume, flow rate, columnpressure, flow-through fraction size, eluate fraction size, and peakfraction size. Once optima are obtained, the user can design experimentsby creating a series of run conditions for a particular procedure to beperformed on the instrument. Commercially available software packagesthat performed this function can be incorporated, such as MODDE ofUmetrics Inc. (San Jose, Calif., USA) and JMP software of SAS InstituteInc. (Cary, N.C., USA).

A method once configured is run on any system that is available on thenetwork with the appropriate configuration. The software automaticallymaps the fluidics scheme in the method against the devices (i.e., thefluid manipulation components) that are installed on the mounting frameand notifies the user in case of discrepancies. In case of ambiguity orwhen multiple devices of the same type are present, the user is able toassociate the physical location of the device with the correspondinglocation in the fluidics scheme. The user has the option of writingadditional descriptive information to a particular run, of schedulingruns to be performed in sequence, of delaying runs or setting times forruns to be performed, of activating evaluation procedures, and ofrecording run data and results.

System control software. The system control component of the softwaresuite enables the user to start, monitor, and control a run. Monitoringcan include viewing the number of experiments in a queue and theexperiment in progress relative to the queue, the phase and step of themethod that is in progress, the time left for the phase/step tocomplete, the total time for the run to complete, and chromatogramscompleted and in progress. Monitoring can also include extending orstopping a run. As noted above, the system control software can includea fluidics library, which is a library of configured components andreplacement components. The user can select a fluidics scheme from thefluidics library or modify the selected scheme (or create one's own) byadding or substituting components from the replacement componentslibrary. The software can be programmed to indicate when a component isincorporated into a fluidics scheme, to calculate dead volume associatedwith connective tubing, or to allow the user to enter the length of thetubing and then to calculate the dead volume based on the enteredlengths. The software can automatically map the fluidics scheme in themethod against the available devices present and notify the user in caseof discrepancies. In case of ambiguity or when multiple devices of thesame type are present, the user is able to associate the physicallocation of each device to its corresponding location in the fluidicsscheme. During a run, the system control software can display thefluidics scheme flow path and monitor signals and parameters of eachcomponent in the scheme. The software can also be programmed to allowthe user to alter operational parameters such as flow rate, pH, and pHgradient, and to do so directly on the displaced fluidics scheme duringa run. The software can also be programmed to operate a run manually,selecting all run parameters independently of any of the pre-programmedmethods. The chromatogram generated during a manual operation as well asthe sequence of commands used to execute the manual run can be saved.The sequence of commands saved can be replayed as a manual method withor without modification.

Monitoring of a run can include displaying active flow paths throughoutthe performance of the run. This can be achieved with the same LEDs orother indicator lights that are used for the click-and-plumb operation,whether the lights are on the modules themselves or are virtual lightsor tubing segments on a diagram displayed on the computer screen. As inthe use of computer graphics in the click-and-plumb operation, an activeflow path can indicated by the appearance of a line, otherwise absentfrom the display, representing a tubing segment, a change in color orintensity of such a line, or a change from a blinking appearance to acontinuous appearance. Many flow schemes contain multiple pumpingsystems, including for example a two-pump combination arranged toproduce a gradient flow of buffer solution plus a sample pump to prime asample loop or to feed sample directly to a column. The fluid lineleading to or emerging from any one pump will be indicated by indicatorson that line or at the two extremities of the line. When both pumps onthe gradient flow system are in operation, indicators on the outputlines from each pump will show those lines to be in the active mode, andwhen the system progresses from one stage or function of the operationto another, the display will show the change in active flow path. Theindicators will thus enable the operator to see the system's stage ofoperation in real time. In the event of a programming error within themethod, the indicator lights can alert the operator that flow isoccurring in locations where flow was not intended, thereby allowing theoperator to rewrite the program and thereby correct the error. In theevent of a fluid leak during operation, the indicator lights can directthe operator to only those locations where fluid should be flowing. Thiswill help the operator to find the location of the leak.

In addition to the functions described above, the system controlcomponent of the software suite can include a function that displays thepotential fluid paths for pumps that are included in the flow scheme.This function can be used to display the path along which a particularpump when operating would cause fluid to flow, Such information would beuseful in ensuring that fluid will go where intended once the pump isstarted. The indication of an undesired flow path would inform the userof an improper connection having been made, and thereby allow the userto correct the error. This type of indication can be distinguished fromindications representing click-and-plumb operations or active flowpaths, for example, by using different colors, different intensities orline widths, different types of lines (dotted or dashed vs. solid, forexample), or different signals (flashing or blinking vs. steady, forexample). Here again, the indication or signal can be generated by LEDson the modules themselves, or indications on a diagram on the computerscreen, such as virtual LEDs or lines representing tubing segments.

A further function that can be served by indicators on the modules or ona computer screen is to show the locations of ports where manualinjection can be made, either by syringe or other means, and the flowpaths that the fluid will follow once injected through those ports.Manual injection is often used for filling a sample loop or forcalibrating a pH meter, for example. The indicator can help the userselect the correct port for a particular injection.

A still further function that can be served by indicators is to indicatethe positions of rotary valves. A rotary valve will often be rotatablebetween two or more positions that either direct the fluid passingthrough the valve to different flow paths downstream of the valve orthat receive fluid from different sources or incoming lines to thevalve, or both. Rotary valves can thus switch the system between sampleloop priming, sample injection, the flow of an elution buffer solutionthrough a column, and washing the sample loop or other parts of thesystem with wash fluid, and also in many cases stopping flow orrerouting the flow around the valve. Certain users will wish to know theposition of a valve at a particular point in time, either to check tosee if proper connections have been made or to trouble shoot a flowproblem or aberration in the system operation. The position of thevalve, as well as the ports of the valve that are in an active flowpath, can be indicated by any of the methods listed above.

While some or all of the functions listed above can operatesimultaneously by use of distinguishable signals and/or distinguishablechanges in signals, it will be most convenient in certain embodiments ofthe invention include means within the software to allow the user toselect the functions to be displayed.

When a diagram on a computer monitor is used for any of the reasonsdescribed above, the diagram can be a layout of the actual configurationof the system components and flow paths, or an abstract or simplifiedrepresentation of the configuration. Abstract or simplifiedrepresentations are advantageous due to the ease by which they can befollowed. A typical abstract representation arranges the primary flowpath components in a horizontal line with the different componentsrepresented by distinct icons, the components joined by linesrepresenting tubing connections. Boxes above particular components caninclude information relating to the component such as flow rate,temperature, pressure, volume, and other parameters.

The system control software can also be programmed to calibrate modules,such as pumps, conductivity flow cells, pH probes, fraction collectors,and auto-samplers, as well as to record and log all calibration events.Auto-calibration upon start-up can also be an option, including stepwiseinstructions to the user. Diagnostic functions for the variouscomponents and modules can also be included. Components that can senderror signals include air sensors, auto-samplers, fraction collectors,pH probes, UV lamps, conductivity monitors, pumps, and valves. Thenumber of times a component has been used and the length of usage timecan be recorded and compared to the maximum number of uses or length ofuse time which can be included in the data supplied by themicrocontroller associated with the component. A warning signal can thenbe generated by the system control software to instruct the user toreplace or regenerate the component.

Further features that can be included in the system control software area method validation capability and a service log. The method validationcapability can allow the user to view a simulation of the fluidic pathand an animated progression of a run prior to performing an actual run.The service log can record times of actuation and deactuation ofcomponents that are susceptible to aging upon use such as UV lamps, pHprobes, filters, and pump seals. The service log can also contain arecord of servicing events. The system control software can also enablethe user to manually override a programmed method, such as by stoppingor pausing a pump, resuming operation of a stopped or paused pump,keeping a pump operating beyond a programmed operation interval,starting and stopping a fraction collection, proceeding forward to asubsequent step in the protocol, and any of various further commands andoptions within an individual override operation.

(4) Evaluation software. Evaluation software can serve a wide range offunctions, including displaying results, analyzing data, quantifying andcalibrating results, comparing peaks with each other and chromatogramswith other chromatograms, comparing samples, preparing and customizingreports, and importing and exporting data.

The results can for example be in the form of chromatograms that areeither displayed on the GUI or recorded in a printout. Data analysis caninclude such functions as peak integration, measuring, analysis, andidentification, peak mapping, baseline determination, and any otherparameters associated with peaks, valleys, and baselines. Quantificationand calibration can include calculations of area percentages, relativeheights, calibrations for variations in sample size and samplepreparation, curve fitting, and peak origin treatments such as recordingan origin location and placing the origin at a selected location.Performance calculations can include retention times, actual orrelative, number of theoretical plates in absolute terms or per unitcolumn length, column capacity factors, peak symmetry or asymmetryfactors, peak widths, and molecular weight estimations based onretention times on a size-exclusion chromatography (SEC) matrix comparedto standard curves. Chromatogram and peak comparisons can includedifferentiations by color, line quality, or size, and the addition oflegends. Report preparation and customization can include standard andcustom formats and scale adjustments. Importation and exportation ofdata can include choices of spreadsheet formats such as Microsoft EXCEL,WORD, POWERPOINT, and pdf, and the capability of sending or receivingdata from other computers or software.

Methods and runs saved in the database can be searched and retrievedthrough a browser. A search can be filtered so that searches madethrough the method editor can allow users to view the list of existinguser methods or method templates, whereas searches performed through anevaluation editor would allow the user to view saved runs through a listof thumbnails or chromatograms. Multiple runs can be selected in thebrowser, reviewed individually and transferred directly into a “comparerun” field under the evaluation editor through a single mouse click.

FIG. 1A is a representative illustration of the principal components ofone example of a modular chromatography system in accordance with thepresent invention. A series of modules 11 are shown separated from eachother for ease of viewing and poised in front of a mounting frame orbase unit 12 which is connected to a computer 13 containing the softwareplatform that operates the system. Each module 11 has a fluidmanipulation component 14 which includes a fluid input 15 and a fluidoutput 16, both of which are accessible from the front end of themodule. Positioned on the front end of the module itself, or at leastvisible from the front end, are a fluid input connection indicator 17and a fluid output connection indicator 18, to guide the user in makingfluid connections between the modules. Also positioned on the front endof the module, or at least visible from the front end, is an additionalindicator 19 that can serve as a status indicator, data indicatorindicating operational information pertaining to the fluid manipulationcomponent, or an alarm indicator, or individual indicators for each ofthese functions. Alternatively, any of these indicators 17, 18, 19 canalso be on the fluid manipulation component rather than the frontsurface of the module. In the interior of each module or exposed at therear end of the module is a microcontroller 21. The mounting frame 12has a series mounting sites in the form of bays 22 to receive themodules, and a column rack 23 on one side of the frame. Two of the bays(in the upper right corner and the right end of the middle row,respectively) are double-wide to accommodate double-wide modules. Asexplained above, the Figure is merely an example; the number of modulesand bays can vary widely, and the frame can be designed to allowadditional rows to be added. Each bay 22 contains an electricalconnector 24 to send signals to, and receive signals from, themicrocontroller 21 in any module that is inserted in the bay. FIG. 1Bshows the reverse side of one module 11, with the exposedmicrocontroller connection 25 that will contact the connector 24 in eachbay 22 of the base unit. Reservoirs 26 (FIG. 1A) for liquids such aselution buffers and wash liquids are placed in an accessible location,in this case atop the base unit 12. Lengths of tubing, not shown,connect the various fluid manipulation components 14 to each other andto the columns in the column rack 23 as well as the reservoirs 26.

FIG. 2 is a front view of a base unit 31 (mounting frame) that issimilar to the base unit 12 of FIG. 1 except that the base unit of thisFigure includes four rows 32, 33, 34, 35, alternately referred to as“stories,” and the upper two stories 32, 33 are rotatable relative toeach other and to the lower two stories 34, 35. The rotation is about avertical axis 36 passing through the centers of each of the fourstories.

FIG. 3 shows the system topology of an example of a chromatographicsystem with multiple work stations that utilizes features of theinvention. Each workstation corresponds to one set of modules 11 and onebase unit 12 of FIG. 1A (or base unit 31 of FIG. 2), and eachworkstation has its own combination of modules, with the fluidmanipulation components of the modules being sample loading components,sample loops, buffer preparation components, detectors, fractioncollectors, and gradient pumps, as shown. In this multi-workstationsystem, a single computer 41 controls the entire group of workstations.The components and fluidic connections in individual workstations willbe selected by the user from a library supplied with the computerprogram or will be custom designed by the user.

FIG. 4 is an outline of one example of a computer program for a “MethodEditor” in accordance with the invention to be used in selecting,designing, or modifying a fluidics scheme for a single workstation. Asan example, a user or a group member 51 (where access is limited to agroup of designated individuals) can “create” a “method” 53 eitherthrough a “Project Folder” 52 or through a “start” menu, bothincorporated in the computer software. The user is then prompted to savethe “New Method” at any point prior to starting a “Run” 54. When theuser creates a “New Method” 55, the user can define the method settings,which include the parameters to be used for that method in addition todefining the “Method Outline” 56 that is to be run. The “Run” 54 and“Results” 57 for that method can be saved by the user in a databasewhere they would be linked to the “New Method” 55 that was used togenerated them.

A user can define a method in various ways, including the use of (i) aphase library 58 or a template library 59, (ii) a pre-programmed method61, (ii) a protocol 62 previously created and saved by the user, (iv) ascouting method 63, or (v) a “Design of Experiments” (DOE) method 64. Agraphic representation of the method being programmed is displayed,allowing the user to make and view changes to each step of the method inboth graphical and tabular mode. Parameters that can be defined in thedesign of a method can include the following:

Technique: The user can select from a list of common chromatographytechniques, such as anion exchange, cation exchange, mixed mode ionexchange, size exclusion, and hydrophobic interaction chromatography.These options can be listed under a “Technique” heading. The softwarecan also permit the user to add new techniques if needed. For fluidicdiagrams requiring multiple columns, the user can select the column forwhich to set the parameters.

Column type: This enables the user to select from a list of options forcolumns from a column library, including competitive columns indifferent column sizes corresponding to the technique selected. Columnswith RFID tags can insert setup information from the tags into thefluidics diagram. For fluidic diagrams requiring multiple columns, theuser can assign each column type to its appropriate position in thediagram. The software can supply default parameters such as pressurelimits, flow rates, etc., based on the column selected, modifiable bythe user. The software can also provide guidance, such as connector typeand part numbers.

Maximum Pressure: A recommended pressure limit that is slightly below(by 1% or a user-defined amount) the maximum pressure of the column willbe displayed. The user then has the option to enter a value differentfrom the recommended value. For values higher than the maximumrecommended pressure, a “caution” prompt will appear, asking the user toconfirm if the value entered is correct. The system can then modulatethe flow rate once the pressure threshold has been reached in order tomaintain the pressure at or slightly below the set pressure threshold.

Minimum flow rate: The minimum flow rate capacity of the pump can be setas a default minimum flow rate for a column fed by the pump. The usercan then have the option of entering a value different from the default.The user can also have the option of selecting the flow rate units.

Elution parameter: The user can program methods in any flow mode, suchas number of column volumes, total volume, or time. A method created fora small column should scale automatically to maintain the same linearflow rate when a larger column is used for the same method.

Buffer Blending: This feature will enable the user to dilute a buffer,or to define either a single point pH or a pH gradient and durationduring an elution and select a buffer to achieve the desired pH or pHgradient.

The phase library 58 enables the user to create a “Method Outline” 56consisting of a sequence of phases such as equilibration, sampleapplication/collection, column wash, elution, column preparation, systempreparation, and column performance test. The user can “drag-and-drop”groups of individual phases from the phase library 58 into the methodoutline, and each phase may be used once or multiple times in a methodoutline. Custom phases can also be created and added to the phaselibrary. The template library 59 is a library of templates, i.e., groupsof phases that are frequently used together. Parameters for each phasewithin a template can be modified by the user once the template is addedto a method. The steps and parameters being used within each phase aredisplayed, and the default parameter values can be altered if desired.Default flow rates or column volumes in the phases would be set based onthe type of column defined by the user. For a column not included in thelibrary, the user can enter the parameters manually.

Equilibration: This phase equilibrates the column before purification orre-equilibrates the column after purification. The user can define theconcentration and volume of buffer to be used for equilibrating thecolumn. Equilibration can also be set to proceed until a certaindetector condition such as a threshold or stability of detection(conductivity, UV, or pH) has been achieved.

Sample Application: The phase applies sample to the column. For sampleapplication, the user selects the sample injection technique, forexample manual injection, injection by use of an auxiliary sample pumpeither through a sample loop or directly onto the column, or injectionby the use of an autosampler with the user specifying the loop or vialtype and volume, the sample volume, and the buffer used for sampleinjection. For sample collection, the user can define how the fractionsare collected, either with the use of a fraction collector, outletvalves, or waste. Further options include the fraction destination,i.e., the type of rack or the vial or well position where the fractionsare to be collected, the collection pattern (S vs. Z), and whetherfraction collection is to be based on fixed volume, peak fractions, or acombination of the two. Peak fractions can be based on peak slope and/orthreshold. For thresholds, the start and end thresholds can be selected.Software can be used that can combine slope and threshold to determinewhen a peak is ascending or descending, and that can detect valleys,peaks, and peak maxima to ignore noise in the chromatogram. For multiplesample injections and sequential runs, the software can notify the userwhen the total collection volume is greater than the total elutionvolume of all the experiments added. Software can also be programmed totrack peaks to the fraction in the tube. Peak fractions can be displayedby selecting a peak on the chromatogram or selecting a fraction tube.The selected peak and the corresponding tube containing that fractionshould be easily identifiable such as by color.

Column Wash: This phase washes the unbound sample from the column aftersample application or removes strongly bound proteins after elution.Parameters that can be defined for this phase include flow rate, ifdifferent from the flow rate in the method setting, wash buffer, andlength of wash. The length of wash can be defined either in columnvolumes or until a certain detector condition is met, such as a stableconductivity or a pH or UV value.

Elution: This phase elutes the sample from the column. Parameters to bedefined include the elution and fractionation settings. Elutiontechniques can be selected from the following options: (a) isocratic(i.e., elution with a constant eluent composition, which is commonly thesame composition as that of the solution used to equilibrate thecolumn), in which the length of elution (in terms of the number ofcolumn volumes, volumetric flow rate, time, or volume) can be selectedand a delay for fraction collection can be set if needed; (b) continuousgradient, including linear, concave, and convex gradients, in which thegradient concentration, length, and slope can be selected for lineargradients, and the gradient shape for concave and convex gradients; and(c) step gradient, in which one or more steps can be selected from apull-down menu. An option to clean the column after elution with aselected volume and concentration of buffer, or to re-equilibrate thecolumn after elution, or both, can be included, using conditionsprogrammed in at the equilibration step. Step gradients can either bebasic or advanced, in which the buffer concentration and length of thestep in each elution segment can be individually selected. An option canalso be included to flush the system with a set concentration and volumeof buffer between steps and to vary the fraction volume within eachelution segment.

Column Preparation: This phase prepares the column before use byremoving any storage solution present in the column and equilibratingthe column. The inlet position, buffer solution, volume, flow rate andincubation time are selected and the column is filled with buffersolution.

Column Clean-in-Place: This phase cleans the column after purificationruns by rinsing the column with a cleaning solution to removenonspecifically bound proteins. The inlet position, the cleaningsolution volume, flow rate and incubation time are selected and thecolumn is filled with a cleaning solution. For a column equipped with anRFID tag that records column pressure, a prompt will appear on thedisplay when the column pressure becomes excessively high, instructingthe user to perform Column Clean-in-Place. The Column Preparation phaseand the Column Clean-in-Place phase can both be run either separatelyfrom any run or as phases within a run.

System Preparation: The System Preparation phase removes storagesolution from the system tubing and inlet and fills the tubing and inletwith buffer solution before a run. The inlets, outlets and columnpositions to be prepared are selected and the system is filled with theappropriate buffer solution, with one buffer solution per phase.

System Clean-in-Place: The System Clean-in-Place phase cleans the systemafter purification runs by rinsing the system with cleaning solution.The inlets, outlets and column positions to be cleaned are selected andfilled with the cleaning solution, with one cleaning system per phase.Three System Clean-in-Place phases, for example, can be included toallow three different cleaning solutions to be used. The SystemPreparation and System Clean-in-Place phases can both be run eitherseparately from any run or as phases within a run.

Column Performance Test: The efficiency of a packed column in terms ofheight equivalent to a theoretical plate (HETP) and the peak asymmetryfactor (As) are tested in this phase. After equilibration of the column,sample is injected via a capillary loop and eluted isocratically. Anon-adsorbing sample such as acetone or salt can be used. After theelution, the HETP and As are calculated in an evaluation module.

Referring again to FIG. 4, the system can be equipped withpre-programmed methods 61 from which the user can choose, rather thancreating a new method 55. Examples of pre-programmed methods, each ofwhich can be accompanied by a buffer recommendation, are as follows.

Affinity Chromatography: After equilibration of the column and sampleapplication, the protein of interest is adsorbed to the column ligand.The column is then washed to remove unadsorbed proteins and elution isperformed either by using a buffer containing a competitor to displacethe protein of interest, or by changing the pH or ionic strength.Elution in affinity chromatography is most often performed by asingle-step elution, but can also be performed in two steps, the firstto remove weakly bound material, which is often discarded, and thesecond to remove more strongly bound material. With either type ofelution, the column is then re-equilibrated with start buffer.

Anion Exchange Chromatography: After equilibration of the column andsample application, negatively charged proteins are adsorbed to thecolumn ligand. The column is then washed to remove unadsorbed proteinsand elution is performed either isocratically or by using a gradient ofincreasing salt concentration, the gradient being either a step gradientor a continuous gradient. The column is then washed and re-equilibratedwith start buffer.

Cation Exchange Chromatography: After equilibration of the column andsample application, positively charged proteins are adsorbed to thecolumn ligand. The column is then washed to remove unadsorbed proteinsand elution is performed either isocratically or by using a gradient,stepwise or continuous, of increasing salt concentration. The column isthen washed and re-equilibrated with start buffer.

Chromatofocusing: After equilibration of the column and sampleapplication, elution is performed using a pH gradient, causing theproteins to separate and elute according to their isoelectric points.The column is then re-equilibrated.

Column Clean-in-Place: The column is filled with a cleaning solution,inlet positions are selected, and the solution identity, volume, flowrate and incubation time are entered.

Column Performance Test: After equilibration of the column, anon-adsorbing sample such as acetone or salt is injected via a capillaryloop and eluted isocratically. Values for HETP and As are thendetermined.

Column Preparation: The column is filled with buffer solution, inletpositions are selected, and the solution identity, volume, flow rate,and incubation time are entered.

Desalting: After equilibration and sample application, the proteins areeluted isocratically. This technique is commonly used for bufferexchange.

Gel Permeation Chromatography: After equilibration and sampleapplication, proteins are separated and eluted according to size.

Hydrophobic Interaction Chromatography: After equilibration of thecolumn and sample application using a buffer containing a high saltconcentration, for example 2M (NH₄)₂SO₄, hydrophobic proteins areadsorbed to the column ligand. The column is then washed to removeunadsorbed proteins and elution is performed either isocratically orusing a gradient, stepwise or continuous, of decreasing saltconcentration. The column is then washed and re-equilibrated with startbuffer.

Reverse Phase Chromatography: After equilibration of the column andsample application, hydrophobic proteins adsorb to the column ligand.The column is then washed to remove unadsorbed proteins and elution isperformed either isocratically or by using a gradient, stepwise orcontinuous, of a non-polar organic solvent such as acetonitrile. Thecolumn is then washed and re-equilibrated with start buffer.

System Clean-in-Place: The system is filled with cleaning solution andinlets, outlets and column positions are selected for cleaning. ThreeSystem Clean-in-Place phases for example can be included to allow theuse of three different cleaning solutions.

System Preparation: The system is filled with preparation solution andinlets, outlets and column positions are selected for preparation. TwoSystem Preparation phases for example can be included to allow the useof two different preparation solutions.

The option of using one of various existing methods 61 as shown in FIG.4 allows the user to access methods previously run on the system. Oncean existing method is selected, the user will able to save the methodunder a new name, either after having modified the method or using themethod without modifications.

The scouting method 63 option allows the user to create a set of methodsin which one parameter is systematically varied and to run the methodsin sequence. Such scouting can be used for example to screen columns, tofind an optimal pH, to test a column capacity in terms of sample volume,to optimize gradient length and slope, or to optimize step gradients. Apull-down menu can list different variables from which the user canselect, as well as the number of runs for the scouting experiment.Examples of scouting variables are column volume, flow rate, columnpressure limit, flow-through fraction size, eluate fraction size, peakfraction size, and linear concentration gradient and length.

A system that includes a “Design of Experiments” (DOE) software package64 allows the user to vary multiple parameters at once and create aseries of run conditions for a particular chromatography technique. Theoutput can be imported into a DOE method synthesizer that willautomatically output a series of experiments to verify the DOE designspace. The user can be given the option to select the final number ofexperiments to be run and then execute the runs and produce a reportthat correlates the design output to the experimentally obtainedresults. Commercial DOE software packages such as MODDE and JMP(referenced above) can be used and integrated into the method editor sothat the output from the DOE is generated into an executable method onthe system platform.

FIGS. 5 and 6 are examples of graphical displays that can be used torepresent flow paths and to indicate some of the operations andfunctions described above. Although the Figures are not in color,different colors or different color intensities can be used on thedisplay to differentiate among active and inactive flow paths,pump-driven and manually drive flow paths, principal vs. bypass flowpaths, and other operational information. Flow sensors can be installedon individual tubing segments on or between the modules themselves or onthe modules themselves to confirm that the display is a real-timerepresentation of the actual system. In each of these Figures, thecomponents are shown in a generally linear arrangement with flow fromleft to right, each component is represented by an icon with the linesbetween the icons representing connective tubing, and a legend aboveeach icon lists information pertaining to the component, such as itscapacity, operating parameters, and current status. Certain fluid lines,such as lines leading to bottles of solutions, are eliminated tosimplify the diagrams.

FIG. 5 depicts a flow path that begins with a gradient pump system 71that includes individual pumps 72, 73 for buffers A and B, respectively,and a mixer 74. The legend 75 for the pump states the total flow rate,the pump pressure, and the proportion of buffer B as a volume percent ofthe combined flow at any point in time. Immediately downstream of thegradient pump system 71 is a rotary valve 76 to which are connected amanually operated syringe 77, a sample loop 78, and a waste reservoir 79for excess sample during filling of the sample loop. Immediatelydownstream of the rotary valve 76 is a chromatographic column 81, whoseoutlet leads directly to a detector system 82 that includes a UVdetector 83 and a conductivity detector 84. The legend 85 for thedetector system 82 states the wavelength at which absorbance is detectedby the UV detector and the absorbance being detected at any point intime, the conductance being detected in millisiemens per centimeter, andthe temperature. Downstream of the detector system 82 is a fractioncollector 86, and its associated legend 87 which states for any point intime the rack and tube in use at that time, the volume collected in thattube, and the number of tubes remaining unfilled. The final componentshown is a waste reservoir 88 for fluids passing through the column 81that are not diverted to the fraction collector 86.

One display scheme that can be used with the display of FIG. 5 is toshow all connecting lines in a light or dull color until they are activewhen they change from dull to a bright intensity of the same color. Thesyringe 77 in this example can be shown in a different color, and anylines that are active when the syringe is in operation can likewise beshown in the same color as the syringe.

FIG. 6 depicts a flow path that includes more options than that of FIG.5. The gradient pump system 91 in this scheme is shown with feed linesthat include a four-way valve 92 to allow up to four different solutionsto be blended together in any desired ratio. Between the four-way valve92 and the gradient pump system 91 are two rotary valves 93, 94. Thecommon port of one rotary valve 93 is connected to one pump 95 while thecommon port of the other rotary valve 94 is connected to a second pump96, while port 1 of each rotary valve is connected to the outlet of thefour-way valve 92. The two rotary valves 93, 94 allow each pump todeliver different solutions that are connected by fluid lines to any ofthe multiple ports on these valves. As stated above, neither bottles ofsolutions nor fluid lines from bottles of solutions to the inlets of thevalves 93, 94 are shown. A sample injection system 101 includes a samplepump 102 in addition to the manual syringe 103, sample loop 104, andwaste reservoir 105. The chromatographic column 106 is connected to acolumn selector valve 107 which can be used to select between multiplecolumns, only one of which is shown in this diagram, or to bypass allcolumns. In the diagram, the letters “By” when displayed indicate thatthe valve 107 is currently in the bypass position. A pH detector 109 ispositioned between the UV/conductivity detector 108 and the fractioncollector 110. Legends are associated with each component as in FIG. 5.The display scheme for the flow path of FIG. 6 can use different colors(blue and green, for example) to differentiate between the flow paths ofthe sample injection system 101 and the principal flow path extendingfrom the gradient pump 91 through the column selector valve 107,detectors 108, 109, and fraction collector 110, with bright vs. dullcolor intensities to differentiate between active and inactive flowpaths, and the color grey to indicate bypass routes. Other colors andmethods of differentiation, such as dotted vs. solid lines and blinkingvs. continuous lines, can be used instead or in addition to those statesabove, as will be readily apparent to those of skill in the art ofcomputer displays.

In the claims appended hereto, the term “a” or “an” is intended to mean“one or more.” The term “comprise” and variations thereof such as“comprises” and “comprising,” when preceding the recitation of a step oran element, are intended to mean that the addition of further steps orelements is optional and not excluded. All patents, patent applications,and other published reference materials cited in this specification arehereby incorporated herein by reference in their entirety. Anydiscrepancy between any reference material cited herein or any prior artin general and an explicit teaching of this specification is intended tobe resolved in favor of the teaching in this specification. Thisincludes any discrepancy between an art-understood definition of a wordor phrase and a definition explicitly provided in this specification ofthe same word or phrase.

What is claimed is:
 1. A system for joining a plurality of fluidmanipulation components into a flow scheme for directing fluids to andfrom a chromatographic separation device and for operating said joinedfluid manipulation components according to a selected protocol, saidsystem comprising: a plurality of modules, each said module comprising(a) one of said fluid manipulation components, and (b) a microcontrollerthat transmits operational signals to said fluid manipulation componentin response to commands received from outside said module; a mountingframe comprising a plurality of bays, each of the plurality of baysconstructed to receive a single module, and each of the plurality ofbays comprising a signal connector that couples to and communicates withthe microcontroller of a module mounted in said bay, wherein at leastsome of the bays are constructed to receive single modulesinterchangeably with other said modules; and a software platform incommunication with the microcontroller of each module, said softwarebeing programmed to (1) receive from each of the plurality of modulesinformation identifying a type of fluid manipulation component in saidmodule, (2) recognize from the received information the types of thefluid manipulation components in the plurality of modules, (3)automatically map the plurality of fluid manipulation components to theflow scheme based on the received data, (4) provide to a user of thesystem visual indications where fluid connections to the plurality ofmodules are needed in order to implement said flow scheme using theplurality of modules, and (5) cause said fluid manipulation components,once joined, to direct fluids to and from said chromatographicseparation device in accordance with said protocol.
 2. The system ofclaim 1, wherein each said microcontroller further detects malfunctionsof said fluid manipulation component and emits an alarm signal inresponse to said malfunction.
 3. The system of claim 1, furthercomprising a monitor that graphically displays fluid connections betweenthe fluid manipulation components in accordance with said flow scheme.4. The system of claim 3, wherein the system indicates on the pluralityof modules where the fluid connections are needed in order to implementsaid flow scheme using the plurality of modules.
 5. The system of claim4, wherein each said module further comprises indicator lights for thefluid connections and wherein the system indicates using the indicatorlights where the fluid connections are needed, the indicator lightsbeing responsive to said software.
 6. The system of claim 1, whereineach said module further comprises a component status indicatorindicating operational status of the fluid manipulation component onsaid module.
 7. The system of claim 1, wherein each said module furthercomprises a visual alarm indicator and said alarm signal produces avisually observable change in said alarm indicator.
 8. The system ofclaim 1, wherein said mounting frame further comprises a column rack forholding a chromatographic column.
 9. The system of claim 1, wherein saidsoftware comprises a software suite that includes a library of one ormore flow scheme phases selected from the group consisting ofequilibration, sample application, column activation, column washing,elution, column preparation, and tests of column performance, and saidsoftware is further programmed to enable a user to create a flow schemeusing phases selected from from said library.
 10. The system of claim 1,further comprising a monitor that graphically displays fluid connectionsbetween the fluid manipulation components in accordance with said flowscheme, wherein said software comprises a software suite that comprisesmeans for monitoring a run, being performed on said system using thefluid manipulation components, by displaying on said monitor all activeflow paths during said run.
 11. The system of claim 10, wherein saidsoftware suite further comprises means for displaying chromatographicdata produced by a run performed on said system.
 12. The system of claim11, wherein said chromatographic data is one or more members selectedfrom the group consisting of peak integration, peak measuring, peakidentification, peak mapping, and baseline determination.
 13. The systemof claim 1, wherein said fluid manipulation components include at leastone each of valves, pumps, mixers, sample loops, detectors, and fractioncollectors.
 14. The system of claim 1, wherein said machine-readabledata characterizing said fluid manipulation component is dataidentifying said fluid manipulation component as a member selected fromthe group consisting of a valve, a pump, a mixer, a sample loop, adetector, and a fraction collector.
 15. The system of claim 1, whereinsaid machine-readable data characterizing said fluid manipulationcomponent includes data representative of an operational status of saidfluid manipulation component.
 16. The system of claim 1, wherein saidmicrocontrollers contain radio-frequency identification tags encodingsaid data characterizing said fluid manipulation component.
 17. A systemfor operating a plurality of workstations, each said workstationcomprising a plurality of fluid manipulation components joined in a flowscheme for directing fluids to and from a chromatographic separationdevice and for operating said flow scheme according to a selectedprotocol, each workstation operating independently of all other saidworkstations, and each workstation comprising a plurality of modules andsoftware in accordance with claim 1, and wherein said software for allsaid workstations are consolidated in a single computer.
 18. A computersystem for configuring and controlling a chromatography system, thecomputer system comprising: a graphical display; an input device; acommunication interface; a processor; and software that when executedcauses the computer system to: receive a specification of achromatography flow scheme having a number of fluid connections; receivevia the communication interface data from a number of components of thechromatography system, the data identifying and indicating a type ofeach of the components; map the identified fluid manipulation componentsto the chromatography flow scheme; and display a representation of theflow scheme on the graphical display; wherein each of the components ismounted in a respective mounting site of a mounting frame, each mountingsite comprising a signal connector that couples to the component andthrough which the commands are communicated from the computer system tothe component.
 19. The computer system of claim 18, wherein the softwarefurther causes the computer system to: sequentially receive via theinput device indications of particular ones of the fluid connectionsbetween respective pairs of the components; and guide a user of thesystem in making the fluid connections.
 20. The computer system of claim18, wherein the software further causes the computer system to:recognize that two of the components are of the same type; and prompt auser of the computer system to assign one of the two components of thesame type to a particular location in the flow scheme.